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Differential Roles of Two Major Brain Structures, Mushroom Bodies and Central Complex, for Drosophila Male

Differential Roles of Two Major Brain Structures, Mushroom Bodies and Central Complex, for Drosophila Male Courtship Behavior

Takaomi Sakai, Toshihiro Kitamoto

Department of Anesthesia, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242

Received 12 August 2005; accepted 4 January 2006

ABSTRACT: Drosophila male courtship is a com- plex and robust behavior, the potential for which is ge- netically built into specific neural circuits in the central nervous system. Previous studies using male-female mosaics and the flies with defects in particular brain structures implicated the critical central regions in- volved in male courtship behavior. However, their acute physiological roles in courtship regulation still largely remain unknown. Using the temperature-sensitive Dynamin mutation, shibire ts1 , here we demonstrate the significance of two major brain structures, the mush- room bodies and the central complex, in experience-in- dependent aspects of male courtship. We show that blocking of synaptic transmission in the mushroom body intrinsic neurons significantly delays courtship initiation and reduces the courtship activity by shortening the courtship bout length when virgin females are used as a sexual target. Interestingly, however, the same treat-

ment affects neither initiation nor maintenance of court- ship toward young males that release courtship-stimu- lating pheromones different from those of virgin females. In contrast, blocking of synaptic transmission in a central complex substructure, the fan-shaped body, slightly but significantly reduces courtship activity to- ward both virgin females and young males with little effect on courtship initiation. Taken together, our results indicate that the neuronal activity in the mush- room bodies plays an important role in responding to female-specific sex pheromones that stimulate initiation and maintenance of male courtship behavior, whereas the fan-shaped body neurons are involved in mainte- nance of male courtship regardless of the nature of

courtship-stimulating cues. ' 2006 Wiley Periodicals, Inc. J Neurobiol 66: 821–834, 2006

Keywords: Drosophila melanogaster; courtship; mush- room body; central complex; shibire

INTRODUCTION

Male courtship is arguably the most complex behav- ior performed by the fruit fly Drosophila melano- gaster. It is a stereotyped sequence of behavioral pat-

Correspondence to: T. Kitamoto (toshi-kitamoto@uiowa.edu). Contract grant sponsor: Uehara Memorial Foundation (T.S.). Contract grant sponsor: NIH; contract grant number: MH62684 (T.K.). This article includes Supplementary Material available via the Internet at http://www.interscience.wiley.com/jpages/0022-3034/ suppmat ' 2006 Wiley Periodicals, Inc. Published online 3 May 2006 in Wiley InterScience (www.interscience. wiley.com). DOI 10.1002/neu.20262

terns, composed of orientation, tapping, wing exten- sion and vibration, licking, attempted copulation, and copulation (Bastock and Manning, 1955; Cobb et al., 1986; Welbergen et al., 1987). Multiple sensory modalities, in particular chemical perception and vision, play important roles in the regulation of dif- ferent behavioral elements in male courtship (Hall, 1994; Greenspan and Ferveur, 2000). The main pher- omone components of male courtship are cuticular hydrocarbons (Jallon, 1984; Ferveur and Jallon, 1996), which are mainly nonvolatile and detected by specific gustatory neurons (Bray and Amrein, 2003). Volatile pheromones emitted by females also stimu- late male courtship (Tompkins et al., 1980; Tomp- kins, 1984). In addition, dynamic as well as static vis-

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822 Sakai and Kitamoto

ual stimuli are needed for efficient courtship in D. melanogaster (Markow and Manning, 1980; Tomp- kins, 1984; Hall, 1994; Sakai et al., 1997, 2002). Because a male fly kept in social isolation is fully capable of carrying out the entire courtship in response to the aphrodisiac sensory cues, the potential for this behavior must be genetically built in the cen- tral nervous system as the particular neural circuits and the molecular machinery regulating their physiol- ogy (Hotta and Benzer, 1976; Hall, 1979; Baker et al., 2001). To understand how courtship behavior is controlled by the nervous system, we need to iden- tify the components of the relevant neural circuits in the brain and elucidate how they interpret the multi- ple sensory cues and regulate the highly coordinated motor outputs. The mushroom bodies (MBs) and the central com- plex (CC) are two prominent structures in the Dro- sophila brain [Fig. 1(A)] that have been extensively studied in terms of their roles in a variety of behav- iors. The MBs are paired structures composed of thousands of intrinsic neurons called Kenyon cells whose cell bodies are localized in the dorso-posterior region of the brain. They receive multimodal sensory information via the dendritic calyx mainly from the olfactory centers of the antennal lobes (ALs) and from other brain regions including the gustatory cen- ters of the subesophageal ganglion (SOG) (Strausfeld et al., 1998; Ito et al., 1998). The Kenyon cells send axonal projections to the anterior part, where they bifurcate to form the medial and vertical lobes. The MBs are considered to be the ‘‘memory center’’, play- ing critical roles in various learning and memory paradigms such as olfactory conditioning, experi- ence-dependent courtship suppression, and context generalization in visual learning (Dubnau et al., 2001; McGuire et al., 2001; McBride et al., 1999; Liu et al., 1999). In addition, nonlearning functions in walking (Martin et al., 1998) and centrophobism/thig- motaxis (Besson and Martin, 2005) are also attributed to the MBs. Another significant structure is the CC, which is located centrally in the brain. It consists of four characteristic neuropils, the protocerebral- bridge, the fan-shaped body [FB; Fig. 1(A)], the ellip- soid body [EB; Fig. 1(A)], and the noduli. The CC forms elaborate connections to a variety of brain regions (Hanesch et al., 1989). Previous studies have indicated the involvement of the CC in the olfactory learning task (Heisenberg et al., 1985), as well as in different features of motor outputs such as mainte- nance or temporal organization of locomotion (Strauss and Heisenberg, 1993; Martin et al., 1999) and visual flight control (Ilius et al., 1994). Further- more, Joiner and Griffith (1999) revealed the involve-

more, Joiner and Griffith (1999) revealed the involve- Figure 1 GAL4 expression patterns in the adult

Figure 1 GAL4 expression patterns in the adult brain of eight GAL4 lines. (A) Schematic representation of mush- room bodies (MBs), ellipsoid body (EB), and fan-shaped body (FB) in adult brain. (B–J) GAL4 expression patterns in the brain of eight GAL4 lines visualized by GFP reporter gene. F1 males between GAL4 females and UAS-GFP males were used. We observed at least four males in each line. (B) OK107. Arrow, pars intercerebralis (PI); arrow- heads, antennal lobes (ALs); asterisk, optic lobe. (C) 30Y. Arrow, subesophageal ganglion (SOG). (D) c772. (E,F) J183. (G) OK348. (H) 104Y. Arrowhead, nerve bundle in lateral areas. (I) c232. (J) c41.

ment of the CC in the CaM kinase-mediated memory formation induced by courtship conditioning. Compared to the aforementioned functions of the MBs and the CC, their acute physiological roles in experience-independent aspects of male courtship behavior are relatively unknown or still controversial.

Previous studies reported that partial feminization of the MBs by ectopically expressing the female form of

the sex-determining gene transformer (tra F ) resulted

in ‘‘bisexual’’ courtship, suggesting that mate dis- crimination is dependent on subsets of the MB intrin- sic neurons (Ferveur et al., 1995; O’Dell et al., 1995). The MB’s role in mate discrimination was to be reconsidered, however, because based on their large- scale analysis of flies expressing tra F in different

regions of the brain, Kido and Ito (2002) found that bisexual behavior was not correlated with the femini- zation of the MBs. Besides mate discrimination, the involvement of the MBs in other aspects of experi- ence-independent courtship behavior also still re- mains elusive. According to Kido and Ito (2002), ab- lation of the MBs by feeding newly hatched larvae with hydroxyurea resulted in significant reduction in

the male courtship activity toward virgin females,

while the similar hydroxyurea treatment did not affect the amount of their courtship in the study of McBride et al. (1999). As for the CC, the functional study re- garding experience-independent aspects of male courtship is much limited. The involvement of the

CC in male courtship was indicated by the mutants

having structural defects in the CC. These mutants were reported to start courtship with the same latent period as wild-type flies, but to show significantly lower copulation success rates, which could be attrib- uted to their abnormal courtship song (Popov et al.,

2003).

Studies of flies with the partially feminized or structurally damaged brain have provided useful in- formation about central regulation of male courtship behavior. Approaches using these flies, however, are

not sufficient to define the neural circuits for court-

ship, because the neurons playing a key role in court- ship are not necessarily sexually dimorphic. In addi- tion, irreversible developmental defects in particular brain regions, induced chemically or genetically, may cause functional abnormalities in other brain regions

that directly or indirectly interact with the damaged

neurons, which makes it difficult to attribute the observed behavioral defects to the initially damaged

brain regions. To circumvent these potential prob- lems, the temperature-sensitive Dynamin mutation

shibire ts1 (shi ts1 ) can be used in combination with

the Gal4/UAS binary expression system to inhibit

neurotransmission in a spatially specific and tempe- rature-dependent manner (Kitamoto, 2001, 2002). Broughton et al. (2004) have recently utilized the UAS-shi ts1 transgene with various Gal4 lines and ana- lyzed the function of different brain regions in acute physiological control of male courtship. They have found that a cluster of cells in the lateral protocere-

Central Control of Male Courtship

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brum (LPR), a higher-order neuropil region located in the lateral part of the brain, plays a critical role in courtship initiation. Here we further expanded the usage of the UAS- shi ts1 transgene and specifically focused on the acute physiological functions of the MBs and the CC in male courtship behavior. We found that blocking of synaptic transmission in the MBs significantly af- fected courtship initiation as well as maintenance when virgin females were used as a sexual target. In- terestingly, the same treatment did not change their courtship behavior toward immature males that have different pheromone profiles from virgin females and elicit strong courtship behavior from sexually ma- tured males (Tompkins et al., 1980; Hall, 1994). In con- trast, blocking of synaptic transmission in a CC sub- structure, the FB, reduces courtship activity slightly but significantly toward both virgin females and young males, with little effect on courtship initiation. These results suggest that synaptic transmission in the MBs plays a significant role in responding to female-specific pheromones that stimulate courtship initiation and maintenance, whereas neurotransmis- sion in the FB is involved in the maintenance of courtship regardless of the nature of courtship-stimu- lating pheromones.

METHODS

Flies

A wild-type strain Canton-S (CS), eight enhancer-trap Gal4 lines, and a UAS-shi ts1 line were used in this study. OK348, J183, c232, 30Y, OK107, and c772 were outcrossed for at least five generations to white flies with Canton-S genetic background. The origin of genetic background in c41 and 104Y is unknown. All flies were raised on glucose-yeast- cornmeal medium at 23 6 18C in a 12:12 h light/dark (LD) cycle (lights on at 8:00 am). Virgin males and females were collected within 6 h after eclosion and maintained in vials until experiments. All experiments were carried out during daytime between 9:00 and 14:00 hours at 24.5 6 0.5 or 30 6 0.58C. The mating activity of D. melanogaster does not change during the period between Zeitgeber Time (ZT) 0 and ZT 9 (Sakai and Ishida, 2001).

Microscopy

Adult male brains and appendages were dissected in PBS and fixed with PBS containing 3.7% formaldehyde for 15 min at room temperature. GFP fluorescence was observed with a confocal microscope (Zeiss 510). Z sec- tions were collected at 2 m intervals and processed to con- struct projections through an extended depth of focus.

824 Sakai and Kitamoto

Recording and Analysis of Male Courtship

A male and a target male or female were placed in an

acrylic plastic observation chamber (15 mm in diameter 3 mm in depth) using a manual aspirator. Courtship behavior was video recorded (DCR-PC300; Sony, Japan) and the courtship latency (CL) and courtship index (CI) were determined. The CL was defined as the period from the initial presentation of a target object to the initiation of courtship behavior, and the CI was defined as the percent- age of time the male spent performing courtship behavior in

a given observation period. We observed male courtship for 10 min or until the moment of copulation.

In the present study, we used three types of target ani-

mals: sexually mature CS females (3 days old), sexually immature CS females (0–24 h old), and newly emerged white males (0–4 h old). GAL4-line females were mated to UAS-shi ts1 males and F1 male progeny (3–4 days old) were used in the experiment. F1 males between GAL4-line females and CS males (3–4 days old) or between CS females and UAS-shi ts1 males (3–4 days old) were the con- trol. We observed 32–56 males in each genotype and dis- carded the data for males that did not show any courtship behavior during the observation period. Experiments were

carried out either at 25 or 308C. For the 308C experiments, experimental males were pretreated at the temperature for 30–50 min.

A courtship bout is defined as a male’s uninterrupted

display of any courtship elements. We measured frequency

of courtship bout and courtship bout duration by observing

courtship of 30 males in each genotype.

Measurement of the Locomotor Activity

The locomotor activity was measured with a Drosophila Activity Monitor IV (TRIKINETICS, Waltham, MA). A male fly (3–4 days old) was introduced into a glass tube (4 mm in diameter and 65 mm in length) through which an infrared beam crossed the path of the fly’s movement. The activity monitor was shaken for 3 s and kept at 25 or 308C for 30 min, followed by the measurement of the locomotor activity for 20 min. The number of incidences at which the

fly crossed the infrared beam was counted every 1 min.

Statistics

In some cases values of the CL, CI, locomotor activity, and

courtship bout duration were not distributed normally.

Thus, we carried out the arcsine transformation of the data

of CI, and the log transformation of the data of the other pa-

rameter. When the transformed data distributed normally and homoscedasticity was evident, such data were analyzed using a two-sample t-test. When the data were not normally distributed after the transformation, they were analyzed using the Mann-Whitney U test. We used computer soft- ware (SPSS 11.5J Base System for Windows) in these tests.

RESULTS

Analysis of Gal4 Expression Patterns Using the Green Fluorescent Protein (GFP) Reporter Gene

In order to examine the physiological functions of the MBs and the CC in male courtship, we used the tem- perature-sensitive Dynamin mutation shi ts1 in combi- nation with eight Gal4 lines that have been reported to specifically mark the MBs or the CC. To confirm and document expression patterns of these Gal4 lines, we visualized Gal4-positive cells in the adult nervous system by driving GFP. Three Gal4 lines, OK107, 30Y, and c772, showed strong GFP expression in the MBs as reported previ- ously (Connolly et al., 1996; Zars et al., 2000; McGuire et al., 2001) [Fig. 1(B–D)]. Besides the MBs they showed scattered GFP expression in the optic lobes [an asterisk in Fig. 1(B)] and restricted, apparently nonoverlapping expression in the ALs [arrowheads in Fig. 1(B)] as well as the SOG [an arrow in Fig. 1(C)]. In these three lines GFP expres- sion was also observed in the pars intercerebralis (PI) located in the median part of the brain [arrow in Fig. 1(B)]. Although weak, GFP expression was de- tected in the EB within the CC for c772 (data not shown). We confirmed that three ‘‘FB’’ Gal4 lines, J183, OK348, and 104Y (Joiner and Griffith, 1999; Con- nolly et al., 1996; Rodan et al., 2002), directed GFP expression in the FB, but not in the EB [Fig. 1(E–H)]. Expression outside the FB was most obvious in J183 [Fig. 1(F)], where the restricted subsets of the MB neurons as well as scattered cells in the optic lobes were GFP-positive [Fig. 1(E)]. OK348 is most spe- cific to the FB among these three Gal4 lines [Fig. 1(G)], although faint expression was detected in the MBs. In addition, a cluster of cells in the lateral region of the brain showed prominent expression [Fig. 1(G)]. In 104Y, GFP expression was also seen in cells in the optic lobes and the SOG as well as nerve bundles in the lateral regions of the brain [an arrowhead in Fig. 1(H)]. GFP expression was not observed in the MBs for this line [Fig. 1(H)]. GFP expression was highly restricted in the EB for c232 [Fig. 1(I)] as reported by O’Dell et al. (1995). c41 showed strong GFP-expression in the EB and weak expression in subsets of the MB neurons [Fig. 1(J)]. GFP-positive cells were also seen in the PI as well as the SOG for c41 [arrow and arrowhead in Fig. 1(J)]. We found that a few Gal4 lines showed GFP expression in the limited groups of putative sensory neurons associated with gustatory receptors in the

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Central Control of Male Courtship 825 Figure 2 Effects of temperature on male courtship behavior. The

Figure 2 Effects of temperature on male courtship behavior. The CL (A,C) and the CI (B,D) were measured at 25 (open bars) and 308C (solid bars). Mature or immature virgin CS females were used as a sexual target. We observed 24 CS males toward mature and immature females, 32 104Y/ þ males toward mature females, and 48 104Y/þ males toward immature females. We discarded the data obtained from males that did not show courtship behavior during the observation period. Error bars show SEM. *p < 0.05. NS, not significant. (A) Mean courtship latency in CS males. n ¼ 23–24 in each bar. (B) Mean courtship index in CS males. n ¼ 23–24 in each bar. (C) Mean court- ship latency in 104Y/þ males (104Y/þ; F1 progeny between 104Y females and CS males). n ¼ 31–43 in each bar. (D) Mean courtship index in 104Y/þ males. n ¼ 31–43 in each bar.

proboscis and the leg (see Supplementary Material). In labial palps of 30Y males, in addition to strong GFP expression in non-neuronal structures called the labellar glands, a subset of gustatory neurons showed positive signals. GFP expression was also observed in restricted putative sensory neurons in the tarsus of the legs for 30Y, OK348, and J183 males. Apparently, the sporadic PNS expression observed in these Gal4 lines did not overlap each other.

Effects of Disrupted Neurotransmission in the MBs and the CC on Male Courtship Behavior toward Virgin Females

To investigate the physiological functions of the MBs and the CC in male courtship, we expressed shi ts1 in these structures using the eight Gal4 lines and examined the effects of conditional disruption of Dynamin function on courtship activity toward virgin females by comparing the relative numbers

for mean courtship latency and courtship index at

25 and 308C. Disruption of Dynamin function in neurons most probably results in acute disruption of neurotransmission. We used immature virgins, instead

of mature virgins, as a female target throughout this study because under the conditions we used, the CIs of control males (CS and 104Y/þ) toward mature vir-

gin females (3–4 days old) were slightly but signi-

ficantly higher at 30 than at 258C [Fig. 2(B,D)]. This is apparently because mature virgins were more receptive at 258C and copulated with males in shorter time with less courtship than at 308C. The CI and

CL toward immature virgin females (0–24 h old),

which stimulate male courtship but do not copulate (Gailey et al., 1984), were not affected by tempera-

ture change.

When shi ts1 expression was directed by OK107, 30Y, and c772 and temperature was raised, the male flies showed a common phenotype in courtship to- ward immature virgins. The latency to initiate court- ship was significantly lengthened and the courtship

826 Sakai and Kitamoto

826 Sakai and Kitamoto Figure 3 Effects of shi t s 1 expression in eight GAL4

Figure 3 Effects of shi ts1 expression in eight GAL4 lines on courtship behavior toward immature virgin females at permissive and restrictive temperatures. Male courtship behavior was observed at 25 (open bars) and 308C (solid bars). Error bars show SEM. NS, not signifi- cant; *p < 0.05; **p < 0.01; ***p < 0.001. (A) Mean courtship latency in three Gal4 lines (OK107, 30Y, and c772) with strong expression in the MBs. n ¼ 32–40 in each bar. (B) Mean courtship index in OK107, 30Y, and c772. n ¼ 32–40 in each bar. (C) Mean courtship la- tency in three Gal4 lines (J183, OK348, and 104Y) with expression in the FB. n ¼ 35–56 in each bar. (D) Mean courtship index in J183, OK348, and 104Y. n ¼ 35–56 in each bar. (E) Mean courtship latency in two Gal4 lines (c232 and c41) with expression in the EB. n ¼ 33– 40 in each bar. (F) Mean courtship index in c232 and c41. n ¼ 33–40 in each bar.

activity was reduced at 308C, compared to those at 258C [Fig. 3(A,B)]. Control males heterozygous for either UAS-shi ts1 [CL at 258C ¼ 60.1 6 11.1 s (n ¼ 32), CL at 308C ¼ 70.0 6 13.3 s (n ¼ 26), Mann- Whitney U ¼ 376, p ¼ 0.53; CI at 258C ¼ 85.1 6 2.2% (n ¼ 32), CI at 308C ¼ 81.6 6 2.6% (n ¼ 26), Mann-Whitney U ¼ 327.5, p ¼ 0.17] or Gal4 insertions showed neither the lengthened CL nor reduced CI at 308C [Fig. 3(A,B)]. Because OK107, 30Y, and c772 shared strong expression in the MBs, but not in other brain regions or PNS neurons, the results suggest that the disruption of synaptic trans- mission in the MBs causes defects in courtship initia- tion and maintenance. The reporter gene expression was observed in PI neurons for all these lines. It is not likely, however, that the PI is responsible for the observed temperature-dependent phenotype, because c41 with expression in the PI did not show the court- ship defects [Fig. 3(E,F)]. Expression of shi ts1 using three Gal4 lines, J183, OK348 and 104Y resulted in the same temperature- dependent courtship phenotype. The CIs showed the slight but statistically significant reduction at 30 compared to at 258C, whereas the CL was not affected by the temperature change [Fig. 3(C,D)]. As shown in Figure 1 these Gal4 lines all showed marked expression in the FB, suggesting that dis- ruption of neurotransmission in the FB results in reduction of courtship activity, but leaves courtship initiation intact. J183 also showed reporter gene expression in the MBs, but contrary to other MB expressing Gal4 lines, the CL was not extended at 308C. The MB expression in J183 was relatively weak and restricted to small subsets of the MB neu- rons. It is possible that J183 does not direct gene expression in the MB neurons that play a role in courtship initiation. Alternatively, the expression level of shi ts1 in the pertinent MB neurons was too low to block synaptic transmission in J183/ UAS- shi ts1 . Similarly, OK348 showed only faint expres- sion in the MBs, therefore the effect of shi ts1 in the MBs might be disregarded. In contrast to other Gal4 lines, c232/UAS-shi ts1 and c41/UAS-shi ts1 males did not show any temp- erature-dependent abnormality in courtship [Fig. 3(E, F)]. In these lines, distinct GAL4 expression was seen in the EB but not in the FB (Fig. 1), implying that disruption of the EB function does not affect male courtship. Although c41 directed Gal4 expres- sion in the MBs, the level of expression was very low [Fig. 1(J)], which might explain why c41/UAS-shi ts1 males did not show defects in the CL and the CI as seen in the Gal4 lines with marked expression in the MBs.

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Central Control of Male Courtship 827 Figure 4 Effects of shi t s 1 expression on

Figure 4 Effects of shi ts1 expression on courtship behavior toward newly emerged males at permissive and restrictive temperatures. Male courtship behavior was observed at 25 (open bars) and 308C (solid bars). Four Gal4 lines, c772, OK107, J183, and 104Y, were used in the exp- eriments. Error bars show SEM. NS, not significant; *p < 0.05. (A) Courtship latency toward immature males. n ¼ 30–39 in each bar. (B) Courtship index toward immature males. n ¼ 27–39 in each bar.

Effects of Disrupted Neurotransmission in the MBs and the CC on Male Courtship Behavior toward Immature Males

In Drosophila, newly emerged immature males have different pheromone profiles from virgin females or mature males, and elicit strong courtship behavior from sexually matured males (Tompkins et al., 1980; Hall, 1994). It is not well known whether courtship behaviors toward virgin females and immature young males require the same critical brain regions. We therefore examined if male courtship toward imma- ture males was affected when neurotransmission in the MBs and the CC was disrupted. In contrast to the earlier result obtained with virgin females as a sexual target [Fig. 3(A,B)], courtship to- ward immature males was not affected by tempera- ture change for the males expressing shi ts1 in the MB neurons using two independent Gal4 lines, c772 and OK107. As shown in Figure 4(A) and (B), the levels of the CL and the CI toward immature males did not show significant differences at 25 and 308C for c772/

UAS-shi ts1 and OK107/UAS-shi ts1 males. This indi- cates that the activity of the MBs neurons is not re- quired for responding to courtship-stimulating cues re- leased from immature males. On the other hand, the temperature-dependent courtship phenotype toward immature males was similar to that toward virgin females when two lines (J183 and 104Y) driving shi ts1 expression in the FB were used. In these flies the higher temperature slightly reduced the courtship ac- tivity with no effect on courtship initiation (Fig. 4). Although J183 expresses the reporter gene in the re- stricted subsets of MB neurons [Fig. 1(E)], the charac- teristic phenotype of J183/UAS-shi ts1 toward immature males is not likely due to the inactivation of these neu- rons because courtship toward immature males did not show any temperature-dependent phenotype in c772 and OK107 directing extensive expression in the MBs. This temperature-dependent courtship phenotype of J183/UAS-shi ts1 and 104Y/UAS-shi ts1 is thus likely to be due to the disruption of neurotransmission in the FB, where J183 and 104Y share expression.

828 Sakai and Kitamoto

Effects of Disrupted Neurotransmission in the MBs and the CC on Courtship Bout Duration

In observations of courtship performed by Gal4 lines expressing shi ts1 in the MBs or the CC, we noticed that it was frequently interrupted and did not last long. To quantify this defect in courtship, we re- corded the duration of each courtship bout performed by 30 males in each genotype at 25 and 308C. The mean duration of courtship bout was calculated from total courtship bouts observed for this analysis. When neurotransmission in the MBs was disrupted at 308C in c772/UAS-shi ts1 males, the frequency of short courtship bouts (<60 s) toward virgin females was strikingly increased and the mean duration of courtship bouts was greatly reduced [Fig. 5(A)]. In contrast, temperatures did not affect mean duration of courtship bouts toward newly emerged males in c772/UAS-shi ts1 [Fig. 5(B)], showing that the effect is specific to courtship toward virgin females. In both c772/þ and þ/UAS-shi ts1 control males, temperature did not significantly affect the mean duration of courtship bouts regardless of the sexual targets [Fig. 5(A,B)]. These results indicate that the on-going ac- tivity of the MB neurons is required for maintaining courtship activity when virgin females are used as a sexual target. J183/UAS-shi ts1 males showed the reduced mean duration of courtship bouts at 308C both toward virgin females and newly emerged immature males [Fig. 5(C,D)], which resulted in reduction of the overall courtship activity regardless of the sexual targets [Figs. 3(D) and 4(B)].

Locomotor Activity at Permissive and Restrictive Temperatures

The general locomotor activity in flies plays an im- portant role in male courtship behavior. Kyriacou (1981) reported that high locomotor activity levels were positively correlated with superiority in male mating performance in D. melanogaster. It is possible that the courtship defects observed in males with functionally disrupted MB or FB neurons are simply due to their low locomotor activity. To address this issue we measured their locomotor activity at 25 and 308C every 1 min for 20 min (see Methods). The locomotor activity was higher at 30 than at 258C in all control males (Gal4/þ and þ/UAS-shi ts1 ) except for c41/þ and 30Y/þ (Fig. 6, solid lines or bars). When shi ts1 expression was directed to the MBs using OK107 and c772, the temperature- induced increase in the locomotor activity was further enhanced in comparison with Gal4/þ and þ/UAS-

shi ts1 controls [Fig. 6(A,B)], indicating that the activ-

ity of the MB neurons normally suppresses general

locomotor activity. This result is consistent with the

report by Martin et al. (1998) demonstrating that chemical ablation of the MBs, a mutation affecting Kenyon cell differentiation (mushroom body minia- ture 1 ), and the disrupted MB neurotransmission all induced the elevated locomotion. As for the Gal4 lines directing expression in the FB, OK348/UAS-

shi ts1 males showed the higher locomotor activity at 30 than at 258C, whereas the levels of the locomotor activity at 30 and 258C were similar in J183/UAS- shi ts1 and 104Y/UAS-shi ts1 males [Fig. 6(C,D)]. In c232 and c41, disruption of neurotransmission in the

EB did not affect locomotor activity [Fig. 6(E,F)]. In

any case, the locomotor activity at 308C was not lower than that measured at 258C for all GAL4 lines expressing shi ts1 . Therefore, it is not likely that the

reduced locomotor activity is a cause of temperature- dependent courtship defects observed in Gal4 lines expressing shi ts1 in the MBs or the FB.

DISCUSSION

In this study we focused on two major structures in

the Drosophila brain, the MBs and the CC, and exam-

ined their physiological roles in experience-independ-

ent aspects of male courtship behavior using the shi ts1

transgene in combination with various Gal4 lines.

Since GAL4 expression is not absolutely limited to

the

MB or CC in ‘‘MB lines’’ or ‘‘CC lines’’ used in

this

study, we cannot formally rule out the possibility

that

neurons outside the MB or CC somehow contrib-

ute

to their shi ts1 -dependent courtship phenotypes.

However, it is most likely that suppression of MB or

CC functions is the main cause of the observed court-

ship phenotypes, because: (1) these GAL4 lines show strong, overlapping GAL4 expression in the MB or CC; (2) all ‘‘MB lines’’ or ‘‘CC lines’’ show, in essence, the same tendency in shi ts1 -dependent court- ship phenotypes; (3) there are no GAL4-positive structures in the brain that are common to the all ‘‘MB lines’’ or ‘‘CC lines’’ other than the MB or CC (except for the PI neurons in the MB lines); and (4) involvement of the MB and CC in male courtship was suggested in other studies. We found that the acute disruption of Dynamin function using the shi ts1 transgene, most probably resulting in blockage of synaptic neurotransmission, in the MBs leads to two clear defects in male court- ship toward virgin females [Fig. 3(A,B)]. First, the length of the period from the initial presentation of a virgin female to the initiation of courtship behavior is

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Central Control of Male Courtship 829 Figure 5 Effects of shi t s 1 expression in

Figure 5 Effects of shi ts1 expression in c772 and J183 on courtship bout duration toward imma- ture virgin females and newly emerged males at permissive and restrictive temperatures. NS, not significant; *p < 0.05; ***p < 0.001. Gal4/þ and þ/UAS-shi ts1 males were used as a control. (A) Histograms and mean duration of courtship bout toward virgin females in c772. (B) Histograms and mean duration of courtship bout toward newly emerged males in c772. (C) Histograms and mean duration of courtship bout toward virgin females in J183. (D) Histograms and mean duration of courtship bout toward newly emerged males in J183.

Figure 6 Journal of Neurobiology. DOI 10.1002/neu

Figure 6

significantly extended (initiation defect). Second, courtship behavior is often interrupted and therefore the length of each courtship bout is shortened (main- tenance defect). Interestingly, these defects are not apparent in courtship toward immature males (Fig. 4). Multiple sensory modalities are integrated to trigger male courtship behavior. Loss of one of the inputs does not abolish courtship, but reduces the intensity and quality of the behavior. Inactivation of the MB neurons is likely to deprive males, at least partially, of the chemical information from virgin females. Our finding suggests that MB-mediated information proc- essing of chemical cues from virgin females plays an important role in courtship initiation as well as main- tenance. Although the MBs are not essential for males to accomplish copulation in laboratory court-

ship settings, it is possible that male flies with defec- tive MBs would be much less fertile in nature because of their subnormal courtship behavior.

In support of our finding, Broughton et al. (2004)

reported that the courtship activity toward virgin females was reduced when synaptic transmission was disrupted using UAS-shi ts1 and the Gal4 lines with strong expression in the MBs. In Broughton et al.

(2004), however, the courtship initiation defect was not detected in males with disrupted MB neurotrans- mission. This apparent discrepancy could be ex- plained by the different experimental conditions between the two studies. The chamber used for assaying court- ship in the current study was larger than the one in Broughton et al. (2004) (15 mm in diameter 3 mm in depth vs. 8 3 mm), resulting in relatively longer courtship latency. In a larger chamber, courtship ini- tiation might be more dependent on the sensory infor- mation processed by the MB neurons, therefore the effect of MB dysfunctions could be more easily detected. In addition, male flies were pretreated at 308C for a longer period in this study (30–50 min vs. 10 min), which may lead to more severe inhibition of the MB neurotransmission.

A short pulse of hydroxyurea administration to

newly hatched larvae results in ablation of the MBs

Central Control of Male Courtship

831

(de Belle and Heisenberg, 1994). As mentioned in the Introduction, the MB ablation by the hydroxyurea treatment was reported to cause a significant decrease in the activity of male courtship in one study (Kido and Ito, 2002), while in the other study the MB abla- tion was reported to have no effect on the quality and quantity of male courtship (McBride et al., 1999). Although it is not known why the similar chemical treatment leads to different behavioral consequences, the possibility exists that the extent of structural defects in the MBs might be somehow different between the two studies. Our observation that block- ing of synaptic transmission in the MB intrinsic neu- rons delays courtship initiation and reduces the court- ship activity rather fits in the result presented by Kido and Ito (2002). On the other hand, a possibility cannot be ruled out at least formally that non-MB neurons could compensate for a role of the MBs in courtship when MB neurons are removed irreversibly during development. If that would be the case, our data would not contradict the result presented by McBride et al. (1999). Heimbeck et al. (2001) found that male courtship was severely impaired when synaptic transmission from the two-thirds of all projection neurons (PNs) was blocked by ectopic expression of tetanus toxin light chain. PNs are the major elements of the central olfactory pathway, which receive information from olfactory receptor neurons in the first-order olfactory neuropils, the ALs (Stocker et al., 1990; Stocker, 1994). PNs transmit the information from the ALs to a higher order neuropil region, the LPR, through two different pathways, either directly without passing through the MBs or indirectly via the MBs (Stocker et al., 1990; Stoker, 1994). Heimbeck et al. (2001) have concluded that the direct neuronal pathway from ALs to the LPR, which is not mediated by the MBs, is necessary and sufficient to process male courtship. However, the argument was based on the assumption that the MBs are not involved in experience-inde- pendent courtship (McBride et al., 1999). Our results indicate that the indirect neuronal pathway from ALs

Figure 6 Effects of shi ts1 expression in eight GAL4 lines on locomotor activity at permissive and restrictive temperatures. The locomotor activity was measured for 20 min at 25 (dotted lines or open bars) and 308C (solid lines or bars). Mean locomotor activity every 1 min and mean total loco- motor activity for 20 min were calculated in each genotype. Gal4/þ and þ/UAS-shi ts1 males were used as a control. Error bars show SEM. NS, not significant; **p < 0.01; ***p < 0.001. (A) Mean locomotor activity every 1 min in UAS-shi ts1 , OK107, 30Y, and c772. n ¼ 42–48. (B) Mean total locomotor activity in UAS-shi ts1 , OK107, 30Y, and c772. n ¼ 42–48. (C) Mean locomotor activity every 1 min in J183, OK348, and 104Y. n ¼ 41–48. (D) Mean total locomotor activity in J183, OK348, and 104Y. n ¼ 41–48. (E) Mean locomotor activity every 1 min in c232 and c41. n ¼ 40– 48. (F) Mean total locomotor activity in c232 and c41. n ¼ 40–48.

832 Sakai and Kitamoto

to the LPR through the MBs also participates in courtship regulation. The fruitless gene (fru) plays a critical role in male courtship behavior. Different fru mutant alleles and their combinations variously disrupt each step of male courtship (Gailey and Hall, 1989; Villella et al., 1997). Recently Demir and Dickson (2005) and Man- oli et al. (2005) have shown that male-specific fru protein (Fru M ), which is expressed in 2% of neu- rons in the male nervous system, is both necessary and sufficient to build the potential for male courtship behavior into the Drosophila nervous system. Stock- inger et al. (2005) and Manoli et al. (2005) have tar- geted the insertion of Gal4 into the fru locus and visualized the neurons expressing male-specific fru protein (Fru M ) and their projections. Many different synaptic neuropils in the brain are labeled for Fru M , including the / -lobe and -lobe of the MBs. Because most, if not all, Fru M -expressing neurons are expected to be involved in male sexual behavior, this is another piece of data indicating the involvement of the MBs in courtship. Compared to the functional disruption of the MBs, that of the CC had different effects on male courtship (Figs. 3–5). Of distinct neuropil regions in the CC, our results indicate that the FB, but not the EB, partici- pates in the proper maintenance of male courtship. The CC has been proposed as a principal site of loco- motor control (Strauss, 2002). Supporting this pro- posal, Martin et al. (1999) showed that continuous blocking of the synaptic transmission by the targeted expression of tetanus toxin in the FB or the EB indu- ces reduction of locomotor activity. However, the slight reduction of the courtship activity observed in male flies with acutely impaired FB neurotransmission cannot be simply explained by a change in locomotor activity, because we could not detect significant reduction in general locomotor activity under the con- ditions where courtship behavior was recorded (Fig. 6). It is not known why the acute disruption of synaptic transmission in the FB leads to reduction of the male courtship activity. One possibility is that the fine reg- ulation of motor outputs, other than the general loco- motor activity, is affected by the disruption of FB neurotransmission, which contributes to the frequent termination of courtship behavior. Alternatively, it is also possible that courtship is defective in the FB- impaired males because the FB neurons integrate sen- sory information and play a role in keeping courtship motivation or interest at a certain state. The reduction of the courtship activity in the FB-impaired males was observed for different sexual targets that release different courtship-stimulating pheromones, implying that the FB controls courtship motivation or interest

at a higher level. In this regard, it should be pointed out that CaMKII activity in the FB is required for the experience-dependent modification of courtship behavior called ‘‘courtship conditioning’’ or ‘‘condi- tioned courtship suppression’’, where a male fly dis- plays a reduced amount of courtship toward virgin females after being paired with a previously mated female (Siegel and Hall, 1979; Joiner and Griffith, 1999). Courtship conditioning is considered to repre- sent an association of an attractive pheromone released by both virgin and mated females and an aversive pheromone released only by mated females (Tomp- kins et al., 1983). An interesting hypothesis is that ex- perience with a previously mated female causes stable physiological changes in the FB of the conditioned male, which in turn leads to the reduction of courtship motivation or interest that lasts for hours and days. Recently we have reported that a circadian clock gene, period, plays a key role in the formation of long-term courtship memory (Sakai et al., 2004). Because the period gene is expressed in the FB (Kaneko and Hall, 2000), per functions might be involved in the possi- ble changes induced in the FB after a male has an ex- perience with a mated female. The synaptic transmis- sion in the FB may play a role not only in experience- independent aspects of courtship but also in its expe- rience-dependent modification.

We thank L. C. Griffith, U. Heberlein, T. Zars, R. L. Davis, M. Saitoe, and J. D. Armstrong for providing GAL4 lines, and M. A. Joiner for useful discussion.

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